Fig 3.

ExsA gain-of-function mutants with altered promoter specificity. (A) Diagram of the nucleotide substitutions in the P_{exoT(C−45A)-lacZ}, P_{exoT(G−47A)-lacZ}, and P_{exoT(A−34C)-lacZ} mutant reporters that were used for the gain-of-function screen. (B to D) The PA103 exsA::Ω strain carrying either the wild-type P_exoT-lacZ reporter or a mutant reporter (P_{exoT(C−45A)-lacZ} [B], P_{exoT(G−47A)-lacZ} [C], or P_{exoT(A−34C)-lacZ} [D]) was transformed with either a vector (V) control (−), an ExsA expression plasmid (wt), or a plasmid expressing ExsA with the indicated amino acid substitutions (K202R, T199S, or T252S). Transformants were cultured under inducing conditions for T3SS gene expression (in panel D, the strains were cultured in the presence of 0.05% arabinose to enhance detection of T252S-dependent activity) and assayed for β-galactosidase activity. The reported values for the β-galactosidase assays (Miller units) throughout this study represent the average of at least three independent experiments, and error bars represent the standard error of the mean (SEM); ** P < 0.01. Anti-ExsA immunoblots demonstrating the steady-state expression levels of ExsA and the K202R, T199S, and T252S mutants are shown as insets.